G protein-coupled receptor kinase-5 attenuates atherosclerosis by regulating receptor tyrosine kinases and 7-transmembrane receptors.

OBJECTIVE: G protein-coupled receptor kinase-5 (GRK5) is a widely expressed Ser/Thr kinase that regulates several atherogenic receptors and may activate or inhibit nuclear factor-κB (NF-κB). This study sought to determine whether and by what mechanisms GRK5 affects atherosclerosis. METHODS AND RESULTS: Grk5(-/-)/Apoe(-/-) mice developed 50% greater aortic atherosclerosis than Apoe(-/-) mice and demonstrated greater proliferation of macrophages and smooth muscle cells (SMCs) in atherosclerotic lesions. In Apoe(-/-) mice, carotid interposition grafts from Grk5(-/-) mice demonstrated greater upregulation of cell adhesion molecules than grafts from wild-type mice and, subsequently, more atherosclerosis. By comparing Grk5(-/-) with wild-type cells, we found that GRK5 desensitized 2 key atherogenic receptor tyrosine kinases: the platelet-derived growth factor receptor-ß in SMCs, by augmenting ubiquitination/degradation; and the colony-stimulating factor-1 receptor (CSF-1R) in macrophages, by reducing CSF-1-induced tyrosyl phosphorylation. GRK5 activity in monocytes also reduced migration promoted by the 7-transmembrane receptor for monocyte chemoattractant protein-1 CC chemokine receptor-2. Whereas GRK5 diminished NF-κB-dependent gene expression in SMCs and endothelial cells, it had no effect on NF-κB activity in macrophages. CONCLUSIONS: GRK5 attenuates atherosclerosis through multiple cell type-specific mechanisms, including reduction of SMC and endothelial cell NF-κB activity and desensitization of receptor-specific signaling through the monocyte CC chemokine receptor-2, macrophage CSF-1R, and the SMC platelet-derived growth factor receptor-ß.

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

Reciprocal regulation of the platelet-derived growth factor receptor-beta and G protein-coupled receptor kinase 5 by cross-phosphorylation: effects on catalysis.

Signaling by the platelet-derived growth factor receptor-beta (PDGFRbeta) is diminished when the PDGFRbeta is phosphorylated on seryl residues by G protein-coupled receptor kinase-5 (GRK5), but mechanisms for GRK5 activation by the PDGFRbeta remain obscure. We therefore tested whether the PDGFRbeta is able to tyrosine-phosphorylate and thereby activate GRK5. Purified GRK5 was tyrosine-phosphorylated by the wild-type PDGFRbeta to a stoichiometry of 0.8 mol phosphate/mol GRK5, an extent approximately 5 times greater than observed with a Y857F PDGFRbeta mutant that fails to phosphorylate exogenous substrates but autophosphorylates and activates Src normally. The degree of PDGFRbeta-mediated phosphorylation of GRK5 correlated with GRK5 activity, as assessed by seryl phosphorylation of the PDGFRbeta in purified protein preparations, in intact cells expressing a tyrosine-to-phenylalanine GRK5 mutant, and in GRK5 peptide phosphorylation assays. However, tyrosyl phosphorylation of GRK5 was not necessary for GRK5-mediated phosphorylation of the beta(2)-adrenergic receptor, even though beta(2)-adrenergic receptor activation promoted tyrosyl phosphorylation of GRK5 in smooth muscle cells. Phosphorylation of the PDGFRbeta by GRK5 in smooth muscle cells or in purified protein preparations reduced PDGFRbeta-mediated peptide phosphorylation. In contrast, phosphorylation of GRK5 by the PDGFRbeta enhanced the V(max) of GRK5-mediated peptide phosphorylation, by 3.4-fold, without altering the GRK5 K(M) for peptide. We conclude that GRK5 tyrosyl phosphorylation is required for the activation of GRK5 by the PDGFRbeta, but not by the beta(2)-adrenergic receptor, and that by activating GRK5, the PDGFRbeta triggers its own desensitization.

Tumor necrosis factor receptor-2 signaling attenuates vein graft neointima formation by promoting endothelial recovery.

OBJECTIVE: Inflammation appears intricately linked to vein graft arterialization. We have previously shown that tumor necrosis factor (TNF) receptor-1 (TNFR1, p55) signaling augments vein graft neointimal hyperplasia (NH) and remodeling through its effects on vascular smooth muscle cells (SMCs). In this study we examined the role of TNFR2 (p75) signaling in vein graft arterialization. METHODS AND RESULTS: Inferior vena cava-to-carotid artery interposition grafting was performed between p75-/- and congenic (C57B1/6J) wild-type (WT) mice. Six weeks postoperatively, neointimal and medial dimensions were greater in p75-/- grafts placed into p75-/- recipients (by 42% or 60%, respectively; P<0.05), when compared with WT veins grafted into WT recipients. Relative to WT vein grafts, p75 deficiency augmented early (2-week-old) graft vascular cell adhesion molecule (VCAM)-1 expression (by 2.4-fold, P<0.05), increased endothelial cell apoptosis (2-fold), and delayed graft re-endothelialization. Both cellular proliferation in early, and collagen I content of mature (6-week-old) vein grafts were increased (by 70% and 50%, respectively) in p75-/- grafts. P75 deficiency augmented TNF-induced apoptosis of cultured endothelial cells, but did not affect TNF-stimulated SMC proliferation or migration induced by co-cultured macrophages. CONCLUSIONS: TNF signaling via p75 reduces vein graft neointimal hyperplasia through mechanisms involving reduction of adhesion molecule expression and endothelial cell apoptosis.

Expression of tumor necrosis factor receptor-1 in arterial wall cells promotes atherosclerosis.

OBJECTIVE: Mechanisms by which tumor necrosis factor-alpha (TNF) contributes to atherosclerosis remain largely obscure. We therefore sought to determine the role of the arterial wall TNF receptor-1 (TNFR1) in atherogenesis. METHODS AND RESULTS: Carotid artery-to-carotid artery interposition grafting was performed with tnfr1-/- and congenic (C57Bl/6) wild-type (WT) mice as graft donors, and congenic chow-fed apolipoprotein E-deficient mice as recipients. Advanced atherosclerotic graft lesions developed within 8 weeks, and had 2-fold greater area in WT than in tnfr1-/- grafts. While the prevalence of specific atheroma cells was equivalent in WT and tnfr1-/- grafts, the overall abundance of cells was substantially greater in WT grafts. WT grafts demonstrated greater MCP-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 expression at both early and late time points, and proliferating cell nuclear antigen expression at early time points. Aortic atherosclerosis was also reduced in 14-month-old apoe(-/-)/tnfr1(-/-) mice, as compared with cognate apoe-/- mice. In coculture with activated macrophages, smooth muscle cells expressing the TNFR1 demonstrated enhanced migration and reduced scavenger receptor activity. CONCLUSIONS: TNFR1 signaling, just in arterial wall cells, contributes to the pathogenesis of atherosclerosis by enhancing arterial wall chemokine and adhesion molecule expression, as well as by augmenting medial smooth muscle cell proliferation and migration.

Regulation of the platelet-derived growth factor receptor-beta by G protein-coupled receptor kinase-5 in vascular smooth muscle cells involves the phosphatase Shp2.

Smooth muscle cell (SMC) proliferation and migration are substantially controlled by the platelet-derived growth factor receptor-beta (PDGFRbeta), which can be regulated by the Ser/Thr kinase G protein-coupled receptor kinase-2 (GRK2). In mouse aortic SMCs, however, we found that prolonged PDGFRbeta activation engendered down-regulation of GRK5, but not GRK2; moreover, GRK5 and PDGFRbeta were coordinately up-regulated in SMCs from atherosclerotic arteries. With SMCs from GRK5 knock-out and cognate wild type mice (five of each), we found that physiologic expression of GRK5 increased PDGF-promoted PDGFRbeta seryl phosphorylation by 3-fold and reduced PDGFRbeta-promoted phosphoinositide hydrolysis, thymidine incorporation, and overall PDGFRbeta tyrosyl phosphorylation by approximately 35%. Physiologic SMC GRK5 activity also increased PDGFRbeta association with the phosphatase Shp2 (8-fold), enhanced phosphorylation of PDGFRbeta Tyr(1009) (the docking site for Shp2), and reduced phosphorylation of PDGFRbeta Tyr(1021). Consistent with having increased PDGFRbeta-associated Shp2 activity, GRK5-expressing SMCs demonstrated greater PDGF-induced Src activation than GRK5-null cells. GRK5-mediated desensitization of PDGFRbeta inositol phosphate signaling was diminished by Shp2 knock-down or impairment of PDGFRbeta/Shp2 association. In contrast to GRK5, physiologic GRK2 activity did not alter PDGFRbeta/Shp2 association. Finally, purified GRK5 effected agonist-dependent seryl phosphorylation of partially purified PDGFRbetas. We conclude that GRK5 mediates the preponderance of PDGF-promoted seryl phosphorylation of the PDGFRbeta in SMCs, and, through mechanisms involving Shp2, desensitizes PDGFRbeta inositol phosphate signaling and enhances PDGFRbeta-triggered Src activation.

Phosphorylation of beta-arrestin2 regulates its function in internalization of beta(2)-adrenergic receptors.

Beta-arrestins mediate agonist-dependent desensitization and internalization of G protein-coupled receptors. Previously, we have shown that phosphorylation of beta-arrestin1 by ERKs at Ser-412 regulates its association with clathrin and its function in promoting clathrin-mediated internalization of the receptor. In this paper we report that beta-arrestin2 is also phosphorylated, predominantly at residues Thr-383 and Ser-361. Isoproterenol stimulation of the beta(2)-adrenergic receptor promotes dephosphorylation of beta-arrestin2. Mutation of beta-arrestin2 phosphorylation sites to aspartic acid decreases the association of beta-arrestin2 with clathrin, thereby reducing its ability to promote internalization of the beta(2)-adrenergic receptor. Its ability to bind and desensitize the beta(2)-adrenergic receptor is, however, unaltered. These results suggest that, analogous to beta-arrestin1, phosphorylation/dephosphorylation of beta-arrestin2 regulates clathrin-mediated internalization of the beta(2)-adrenergic receptor. In contrast to beta-arrestin1, which is phosphorylated by ERK1 and ERK2, phosphorylation of beta-arrestin2 at Thr-383 is shown to be mediated by casein kinase II. Recently, it has been reported that phosphorylation of visual arrestin at Ser-366 prevents its binding to clathrin. Thus it appears that the function of all arrestin family members in mediating internalization of G protein-coupled receptors is regulated by distinct phosphorylation/dephosphorylation mechanisms.

Overexpression of G protein-coupled receptor kinase-2 in smooth muscle cells reduces neointimal hyperplasia.

The activation of vascular smooth muscle cells (SMCs) in neointimal hyperplasia involves signaling through receptor tyrosine kinases as well as G protein-coupled receptors. Overexpression of G protein-coupled receptor kinase-2 (GRK2) in SMCs can attenuate mitogenic signaling and proliferation in response to not only several G protein-coupled receptor agonists, but also platelet-derived growth factor (PDGF). To test whether overexpression of GRK2 could inhibit other SMC responses implicated in neointimal hyperplasia, we assessed SMC chemotaxis and mitogenic signaling evoked by PDGF and G(q)-coupled receptor agonists. To test the effects of GRK2 overexpression on neointimal hyperplasia in vivo, we employed a rabbit autologous vein graft model system. GRK2 overexpression reduced PDGF-promoted SMC chemotaxis by 85% (P<0.01), but had no effect on chemotaxis promoted by epidermal growth factor (EGF). Congruently, GRK2 overexpression reduced by approximately 50% (P<0.05) the [(3)H]thymidine incorporation induced by combinations of PDGF and Gq-coupled receptor agonists, but had no effect on that induced by PDGF plus EGF. PDGF-, but not EGF-promoted phosphoinositide 3-kinase activity in SMCs was also inhibited by GRK2 overexpression. In rabbit vein grafts, we achieved GRK2 overexpression in medial SMCs, reduced cell proliferation during the first week after graft implantation, and reduced steady state neointimal thickness by 29% (P<0.01), without affecting medial thickness or potentiating SMC apoptosis. Because of its ability to dampen chemotactic and mitogenic signaling through PDGF and Gq-coupled receptors, GRK2 overexpression in SMCs may be a useful therapeutic approach for neointimal hyperplasia.

Phosphorylation of the platelet-derived growth factor receptor-beta by G protein-coupled receptor kinase-2 reduces receptor signaling and interaction with the Na(+)/H(+) exchanger regulatory factor.

G protein-coupled receptor kinase-2 (GRK2) can phosphorylate and desensitize the platelet-derived growth factor receptor-beta (PDGFRbeta) in heterologous cellular systems. To determine whether GRK2 regulates the PDGFRbeta in physiologic systems, we examined PDGFRbeta signaling in mouse embryonic fibroblasts from GRK2-null and cognate wild type mice. To discern a mechanism by which GRK2-mediated phosphorylation can desensitize the PDGFRbeta, but not the epidermal growth factor receptor (EGFR), we investigated effects of GRK2-mediated phosphorylation on the association of the PDGFRbeta with the Na(+)/H(+) exchanger regulatory factor (NHERF), a protein shown to potentiate dimerization of the PDGFRbeta, but not the EGFR. Physiologic expression of GRK2 diminished (a) phosphoinositide hydrolysis elicited through the PDGFRbeta but not heterotrimeric G proteins; (b) Akt activation evoked by the PDGFRbeta but not the EGFR; and (c) PDGF-induced tyrosyl phosphorylation of the PDGFRbeta itself. PDGFRbeta desensitization by physiologically expressed GRK2 correlated with a 2.5-fold increase in PDGF-promoted PDGFRbeta seryl phosphorylation. In 293 cells, GRK2 overexpression reduced PDGFRbeta/NHERF association by 60%. This effect was reproduced by S1104D mutation of the PDGFRbeta, which also diminished PDGFRbeta activation and signaling (like the S1104A mutation) to an extent equivalent to that achieved by GRK2-mediated PDGFRbeta phosphorylation. GRK2 overexpression desensitized only the wild type but not the S1104A PDGFRbeta. We conclude that GRK2-mediated PDGFRbeta seryl phosphorylation plays an important role in desensitizing the PDGFRbeta in physiologic systems. Furthermore, this desensitization appears to involve GRK2-mediated phosphorylation of PDGFRbeta Ser(1104), with consequent dissociation of the PDGFRbeta from NHERF.

The adaptor protein beta-arrestin2 enhances endocytosis of the low density lipoprotein receptor.

Endocytosis of the low density lipoprotein (LDL) receptor (LDLR) in coated pits employs the clathrin adaptor protein ARH. Similarly, agonist-dependent endocytosis of heptahelical receptors in coated pits employs the clathrin adaptor beta-arrestin proteins. In mice fed a high fat diet, we found that homozygous deficiency of beta-arrestin2 increased total and LDL plus intermediate-density lipoprotein cholesterol levels by 23 and 53%, respectively (p < 0.05), but had no effect on high density lipoprotein cholesterol levels. We therefore tested whether beta-arrestins could affect the constitutive endocytosis of the LDLR. When overexpressed in cells, beta-arrestin1 and beta-arrestin2 each associated with the LDLR, as judged by co-immunoprecipitation, and augmented LDLR endocytosis by approximately 70%, as judged by uptake of fluorescent LDL. However, physiologic expression levels of only beta-arrestin2, and not beta-arrestin1, enhanced endogenous LDLR endocytosis (by 65%) in stably transfected beta-arrestin1/beta-arrestin2 double-knockout mouse embryonic fibroblasts (MEFs). Concordantly, when RNA interference was used to suppress expression of beta-arrestin2, but not beta-arrestin1, LDLR endocytosis was reduced. Moreover, beta-arrestin2-/- MEFs demonstrated LDLR endocytosis that was 50% less than cognate wild type MEFs. In fusion protein pull-down assays, beta-arrestin2 bound to the LDLR cytoplasmic tail stoichiometrically, and binding was abolished by mutation of LDLR Tyr807 to Ala. Mutation of LDLR cytoplasmic tail Ser833 to Asp enhanced both the affinity of LDLR fusion protein binding to beta-arrestin2, and the efficiency of LDLR endocytosis in cells expressing beta-arrestin2 physiologically. We conclude that beta-arrestin2 can bind to and enhance endocytosis of the LDLR, both in vitro and in vivo, and may thereby influence lipoprotein metabolism.

Phosphorylation of the platelet-derived growth factor receptor-beta and epidermal growth factor receptor by G protein-coupled receptor kinase-2. Mechanisms for selectivity of desensitization.

Accumulating evidence suggests that receptor protein-tyrosine kinases, like the platelet-derived growth factor receptor-beta (PDGFRbeta) and epidermal growth factor receptor (EGFR), may be desensitized by serine/threonine kinases. One such kinase, G protein-coupled receptor kinase-2 (GRK2), is known to mediate agonist-dependent phosphorylation and desensitization of multiple heptahelical receptors. In testing whether GRK2 could phosphorylate and desensitize the PDGFRbeta, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFRbeta in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFRbeta but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFRbeta and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFRbeta mutant, GRK2-mediated PDGFRbeta phosphorylation required receptor tyrosine kinase activity, as does PDGFRbeta ubiquitination. Agonist-induced ubiquitination of the PDGFRbeta, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFRbeta down-regulation. Like the vast majority of GRK2 substrates, the PDGFRbeta, but not the EGFR, activated heterotrimeric G proteins allosterically in membranes from cells expressing physiologic protein levels. We conclude that GRK2 can phosphorylate and desensitize the PDGFRbeta, perhaps through mechanisms related to receptor ubiquitination. Specificity of GRK2 for receptor protein-tyrosine kinases, expressed at physiologic levels, may be determined by the ability of these receptors to activate heterotrimeric G proteins, among other factors.